Once all sister kinetochores have achieved proper bi-oriented attachments, the spindle checkpoint is satisfied. For example, in animals that have a high copy number of mitochondria, transmission of mitochondrial DNA is thought to occur randomly Westermann, On the other hand, a single mitochondrion is present in many unicellular eukaryotes, such as kinetoplastids, Plasmodium falciparum , and Cyanidioschyzon merolae Robinson and Gull, ; Itoh et al.
The timing of duplication and partition of their mitochondria must be coordinated with the cell cycle machinery in these organisms. Kinetoplastids are a group of unicellular organisms that are characterized by the unique structure called the kinetoplast, which is a network of multiple copies of mitochondrial DNA termed the kDNA enclosed in a single mitochondrion Vickerman, They are evolutionarily divergent from commonly studied model eukaryotes e.
Among various kinetoplastids studied thus far, the mechanism of cell cycle is best characterized in Trypanosoma brucei , the causative agent of human African trypanosomiasis for reviews, see McKean, ; Hammarton, ; Vaughan and Gull, ; Li, G1 cells have a single kinetoplast and nucleus termed 1K1N see Fig.
Trypanosomes do not break down their nuclear envelope closed mitosis , and an intranuclear mitotic spindle is assembled in the nucleus during M phase Vickerman and Preston, ; Ogbadoyi et al. Sister kinetochores align at the metaphase plate during metaphase, followed by the separation of nuclear DNA in anaphase creating 2K2N cells and the split of cells by cytokinesis Sherwin and Gull, ; Woodward and Gull, It is essential that replication and segregation of these organelles occur prior to cytokinesis in a coordinated manner so that daughter cells inherit a copy of each.
Little is known about the underlying molecular mechanism. Cyclin B CYC6 is enriched at kinetochores in metaphase and disappears in anaphase. A CYC6 has a dynamic localization pattern during the cell cycle. K and N stands for the kinetoplast and nucleus, respectively. Available evidence suggests that T. For example, when bipolar spindle assembly is blocked in procyclic insect form cells, they undergo cytokinesis without a noticeable delay despite a lack of nuclear division Robinson et al.
This results in the formation of one daughter cell that has one kinetoplast and no nucleus 1K0N, termed zoid and another cell that has one kinetoplast and a nucleus of tetraploid DNA content, suggesting that the spindle checkpoint is not operational Ploubidou et al. In fact, most of the spindle checkpoint components i. Although a Mad2 homolog is present, this protein localizes at basal bodies, not kinetochores Akiyoshi and Gull, It is therefore thought that trypanosomes cannot delay cytokinesis even when nuclear division fails to occur.
Yet, there must be a mechanism to coordinate the segregation of nuclear DNA with cytokinesis in unperturbed cells. One possibility is the presence of a cell cycle oscillator that triggers cell cycle events in a defined sequence even without feedback control systems. The rise and fall of their kinase activities trigger cell cycle events in a set sequence. For example, increased activities of mitotic CDK complexes promote entry into M phase and various mitotic events, whereas their decrease is essential for exit from mitosis.
These observations suggested that degradation of cyclin B could be a trigger for the metaphase-anaphase transition. Here we directly tested this possibility by expressing a non-degradable version of CYC6 in T. We observed the following localization pattern Fig. There was no distinct signal in G1 cells.
From G2 to metaphase, nuclear signal was observed with significant enrichment at kinetochore regions in metaphase. In fact, these nuclear dots co-localized with a kinetochore marker protein, KKT2 Fig. CYC6 disappeared from the nucleus in anaphase. We obtained similar results for CRK3, which formed nuclear dots in metaphase and disappeared in anaphase Fig. Thus, CYC6 and CRK3 exhibit a dynamic localization pattern depending on cell cycle stages, and these proteins can therefore be used as a molecular cell cycle marker.
CDK activities are known to be important for kinetochore assembly in some eukaryotes, including humans Gascoigne and Cheeseman, The finding that CYC6 localizes at kinetochores from G2 to metaphase in trypanosomes prompted us to study its importance for kinetochore assembly.
We confirmed that CYC6 is essential for cell growth Fig. S1 , as previously reported Li and Wang, ; Hammarton et al. We used a spindle marker protein that we identified from our previous tagging screen ORF Tb We observed defective spindle microtubules in CYC6-depleted cells, suggesting that CDK activities are essential for proper bipolar spindle assembly Fig.
Therefore, CYC6 is dispensable for the localization of these kinetochore proteins in procyclic cells. However, we currently do not know whether kinetochores are assembled properly in CYC6-depleted cells. Cyclin B CYC6 is important for bipolar spindle assembly, but dispensable for the localization of many kinetochore proteins.
Note that CYC6-depleted cells have spindle assembly defects, and their chromosomes fail to align onto the metaphase plate. We next used CYC6 as a molecular cell cycle marker to examine the effect of drugs. We first used an anti-microtubule agent, ansamitocin, to test whether bipolar spindle assembly defects affect cell cycle progression Robinson and Gull, After a 4-h treatment, nuclear division and bipolar spindle assembly were perturbed as expected Fig.
In this condition, however, we found no significant enrichment of nuclear CYC6-positive cells Fig. This corroborates previous studies Ploubidou et al. Spindle assembly defects do not prevent cyclin B CYC6 degradation in the nucleus.
A Growth curves of control and ansamitocin-treated cultures show a concentration-dependent growth inhibition BAP B Ansamitocin prevents bipolar spindle assembly. C Ansamitocin treatment does not result in the accumulation of nuclear CYC6-positive cells.
Three hundred cells were counted for each sample, and experiments were performed three times. Error bars represent standard deviation.
P -value was obtained by two-tailed, unpaired t -test. We next examined the effect of cyclin B stabilization for cell cycle progression. We first used a proteasome inhibitor MG that blocked cell cycle progression and stabilized the CYC6 protein Mutomba et al.
Indeed, these cells had a bipolar spindle often elongated and most of their kinetochores were aligned at the metaphase plate Fig. We also noted that the distance between the two kinetoplast DNA in these cells was often greater than that in control metaphase cells. These results suggest that, upon MG treatment, trypanosomes arrest the nucleus in a metaphase-like state in which cyclin B is not degraded, although their cytoplasm transits to an anaphase-like state.
Non-degradable cyclin B CYC6 prevents nuclear division. Then, we analyzed the ubiquitination sites involved in further deg- radation. With this program, the ubiquitination sites of goldfish cyclin B were predicted to be K68, K76, K77 and K The N-terminal region of cyclin B is unfolded and does not contribute to binding with Cdk1 to form the MPF complex27— Thus, it is highly possible that the predicted lysine residues are target sites for ubiquitination.
We next prepared point mutants of all the lysine residues in the lysine-rich stretch following the cut site of cyc- lin B. These findings suggest that some inhibitory mechanisms against the proteasome itself prevent cyclin B deg- radation, at least during metaphase II arrest.
Methods Animals. Xenopus laevis was obtained from a dealer and maintained until use. Xenopus CSF- arrested egg extracts were prepared by the method of Murray et al. All animal experiments were carried out with approval from the Institutional Ethics Committee of Shizuoka University, Japan approval no. Electrophoresis and immunoblot analysis. Electrophoresis proceeded as described by Laemmli34 using Cyclin B degradation was assessed by immunoblotting against anti-goldfish cyclin B B63 monoclonal antibody Production of recombinant cyclin Bs.
Double-strand mutated cDNA was prepared by T3 polymerase using single strand cDNA and the following oligonucleotides containing exchanged nucleotides at the mutation site and a restriction enzyme site, as previ- ously described Mutant clones were screened by proteolytic cleavage with restriction enzymes and confirmed by sequencing. Recombinant proteins were produced in E. Fertilized Xenopus laevis eggs were prepared by in vitro fertilization, as previously described The cell cycle arrest activity of mutant cyclin Bs was assessed after the cleavage of the injected blastomere did or did not stop Uversky, V.
Instrumental analysis of intrinsically disordered proteins: assessing structure and conformation. Wiley, Sheaff, R. Proteasomal turnover of p21 Cip1 does not require p21 Cip1 ubiquitination. Rosenberghasson, Y. Camus, S. Ubiquitin-independent degradation of p53 mediated by high-risk human papillomavirus protein E6. Kalejta, R. Proteasome-dependent, ubiquitin-independent degradation of the Rb family of tumor suppressors by the human cytomegalovirus pp71 protein.
P Natl Acad. Sdek, P. MDM2 promotes proteasome-dependent ubiquitin-independent degradation of retinoblastoma protein. Tokumoto, T.
Initiation of cyclin B degradation by the 26S proteasome upon egg activation. Cell Biol. Roberts, B. Evidence of proteasome-mediated cytochrome P degradation. Alvarez-Castelao, B. Mechanism of direct degradation of IkappaBalpha by 20S proteasome.
FEBS Lett. Kroll, M. The carboxy-terminus of I kappaB alpha determines susceptibility to degradation by the catalytic core of the proteasome. Shaeffer, J. Degradation of Monoubiquitinated Alpha-Globin by 26s Proteasomes. Braten, O. Numerous proteins with unique characteristics are degraded by the 26S proteasome following monoubiquitination.
Natl Acad. Khor, B. Proteasome activator PA is required for normal spermatogenesis. Huang, L. Proteasome activators, PA28 gamma and PA, play indispensable roles in male fertility. Qian, M. Hwang, H. Novel functions of the ubiquitin-independent proteasome system in regulating Xenopus germline development.
Murray, A. Cyclin synthesis and degradation and the embryonic cell cycle. Cell Sci. Figueiredopereira, M. Rock, K. The uptake of proteins into autophagosomes appears to be nonselective, so it results in the eventual slow degradation of long-lived cytoplasmic proteins.
The lysosome system. Lysosomes contain various digestive enzymes, including proteases. Lysosomes take up cellular proteins by fusion with autophagosomes, which are formed by the enclosure of areas of cytoplasm or organelles e.
However, not all protein uptake by lysosomes is nonselective. For example, lysosomes are able to take up and degrade certain cytosolic proteins in a selective manner as a response to cellular starvation.
The proteins degraded by lysosomal proteases under these conditions contain amino acid sequences similar to the broad consensus sequence Lys-Phe-Glu-Arg-Gln, which presumably targets them to lysosomes. A member of the Hsp70 family of molecular chaperones is also required for the lysosomal degradation of these proteins, presumably acting to unfold the polypeptide chains during their transport across the lysosomal membrane.
The proteins susceptible to degradation by this pathway are thought to be normally long-lived but dispensable proteins. Under starvation conditions, these proteins are sacrificed to provide amino acids and energy, allowing some basic metabolic processes to continue.
By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed. Turn recording back on. National Center for Biotechnology Information , U. Show details Cooper GM. Sunderland MA : Sinauer Associates ; Search term. Protein Degradation. The Ubiquitin-Proteasome Pathway The major pathway of selective protein degradation in eukaryotic cells uses ubiquitin as a marker that targets cytosolic and nuclear proteins for rapid proteolysis Figure 7.
Figure 7. Lysosomal Proteolysis The other major pathway of protein degradation in eukaryotic cells involves the uptake of proteins by lysosomes. Cite this Page Cooper GM. In this Page. Recent Activity. Clear Turn Off Turn On. Protein Degradation - The Cell.
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